Investigating a novel Asgard archaeon and its tubulin-like proteins
Since their discovery in 2015, the Asgard Archaea have been redefining our understanding of the origin of the eukaryotic cell. Metagenome-assembled-genomes (MAGs) from various marine environments have shown that this kingdom, officially called Promethearchaeati, encodes many Eukaryotic Signature Proteins (ESPs); proteins that were once thought to be unique but ubiquitous to eukaryotic cells. The presence of ESPs in the Asgard Archaea has challenged the definition of a eukaryote, suggesting that certain ‘eukaryotic-specific’ systems, such as a complex cytoskeleton, may have originated prior to eukaryogenesis (the evolutionary process that gave rise to the Last Eukaryotic Common Ancestor (LECA)). Phylogenetic analyses of the many Asgard archaeal MAGs that have become available suggest a strong affiliation between Asgard Archaea and Eukarya, either as sister lineages or with Eukarya emerging from within the Asgard Archaea phylum, making these Archaea essential for studying the origin of eukaryotes. Moreover, the cytoskeleton is a vital, dynamic network of proteins that regulates cell structure, shape, and division, and our evolutionary understanding of the transition from the prokaryotic to the eukaryotic cytoskeleton remains incomplete. The phenotypic characterisation of the Asgard Archaea and validation of the existence of a eukaryotic-like cytoskeleton has been hampered by the difficulty of culturing and isolating these organisms. To date, only three Asgard archaeal enrichments have been published: Promethearchaeum syntrophicum MK-D1, Candidatus Lokiarchaeum ossiferum B35, and Ca. Harpocratesius repetitus FW102, all of which have been unable to be isolated but are maintained in co-culture with other organisms. This inability to isolate pure cultures has hampered research; therefore, in vitro techniques have been used to biochemically characterise cytoskeletal proteins identified in their genomes. Examples of these proteins include the Asgard actin homologue, lokiactin. Electron microscopy and fluorescence microscopy have shown that a lokiactin-based cytoskeleton exists in Ca. Lokiarchaeum ossiferum, suggesting the origin of the actin cytoskeleton arose prior to eukaryogenesis. At the beginning of this thesis work, the other major cytoskeletal protein, tubulin, remained largely unexplored in the Asgard Archaea. Eukaryotic tubulins are members of the Tubulin Superfamily (TSF), alongside their prokaryotic counterparts, such as FtsZ, and are critical components of the cytoskeleton. In vitro characterisation of the eukaryotic tubulin homologue from Odinarchaeota (called OdinTubulin), demonstrated a mosaic of eukaryotic structural features with prokaryotic biochemical features. However, without a cultured representative, it is not possible to validate and characterise the presence of eukaryotic-like tubulins in vivo. This thesis investigates the TSF proteins from a novel lineage of Asgard Archaea, Nerearchaum marumarumayae Loki-ASV2. The work begins by introducing the Asgard Archaea and the cytoskeletal proteins they encode, the challenges in phenotypically characterising these proteins within the Asgard kingdom, and an overview of the distribution, structure, and function of Tubulin Superfamily proteins across the Tree of Life. The practical work of this thesis has aimed to cultivate and characterise a novel lineage of the Asgard Archaea; to understand the diversity and distribution of TSF proteins in the Asgard Archaea to gain insight into the evolution of TSF proteins; and to biochemically characterise eukaryotic-like tubulin proteins from Loki-ASV2. The thesis concludes by underlining the importance of Loki-ASV2 tubulin proteins as analogues for studying proto-eukaryote tubulins and by highlighting the need for further research into the environmental contexts in which Asgard Archaea are found. Overall, I surmise that the eukaryotic tubulin cytoskeleton is unlikely to have originated in the Asgard Archaea, but that the Asgard tubulin proteins can serve as an analogous protein system for understanding their evolution in a proto-eukaryote. Moreover, I support further investigation of Asgard Archaea in their environmental contexts to elucidate the function of the uncovered surface-structure proteins and the environmental factors that enable horizontal gene transfer in Asgard Archaea.
archaea, eukaryogenesis, microbial evolution, tubulin, anzsrc-for: 310199 Biochemistry and cell biology not elsewhere classified, anzsrc-for: 310799 Microbiology not elsewhere classified, anzsrc-for: 310299 Bioinformatics and computational biology not elsewhere classified
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